Sunflower (Helianthus annuus) oil is a major edible oil worldwide. The oil component of sunflower seeds typically contributes about 80 percent of the value of a sunflower crop and is mostly used as a cooking medium. Sunflower oil also is employed as salad oil, as well as in the manufacture of margarine, soap, and shortening. Shortening is a fat, such as butter or lard, used to make cake or pastry light or flaky. These uses of sunflower oil however, are restricted by the amount of processing needed to modify the fatty acid composition of sunflower oil to eliminate the problems of rancidity, odor and texture.
Because of its high degree of unsaturation, sunflower oil is susceptible to oxidative changes during processing and storage as described, for example, by C. F. Adams, Nutritive Value of American Foods, Agricultural Handbook 456 (U.S. Department of Agriculture, 1975). The stability and flavor of sunflower oil is improved by hydrogenation, which chemically reduces fatty acid double bonds. But the need for this processing reduces the economic attractiveness of sunflower oil.
The principal fatty acids present in sunflower are the diunsaturated fatty acid linoleate, which comprises about 65 percent of the total, and the monounsaturated fatty acid oleate, which comprises about 25 percent of the total. Sunflower oil also comprises smaller amounts of saturated fatty acids, primarily palmitate and stearate. Palmitate constitutes about 4.5 percent to about 6.0 percent and stearate constitutes about 5.0 to about 6.0 percent of sunflower seed fatty acid.
These fatty acids are not present in the plant oil in their free form but primarily are found esterified to glycerol in the form of triglycerides. During plant oil fatty acid composition analysis, the triglycerides typically are broken down to release methyl derivatives of the constituent fatty acids.
Because of its fatty acid composition, unmodified sunflower oil is not well suited for the production of high quality margarines. One way to overcome this problem is to interesterify sunflower oil with another hydrogenated vegetable oil as suggested by Freier et al., Ind. Aliment. (Bucharest) 24: 604-07 (1973). Margarine manufacturers in the United States often interesterify sunflower oil with safflower or soybean oil, and then blend in a portion of hardened hydrogenated oil. Unfortunately, such processing leads to higher costs and health concerns due to the undesirable formation of trans fatty acids. If a sunflower oil contained a greater proportion of palmitate and stearate, then less processing would be needed to make margarine from it.
Oil can be converted into an edible fat for the confectionery industry. Unfortunately, fatty acid composition of conventional sunflower oil prevents the extensive use of this oil in the confectionery industry, for example, as a substitute for cocoa butter.
More than 2 billion pounds of cocoa butter, the most expensive edible oil, are produced annually in the world. The U.S. imports several hundred million dollar's worth of cocoa butter annually. The high prices and uncertain supplies of cocoa butter have encouraged the development of cocoa butter substitutes that have fatty acid compositions similar to cocoa butter.
The fatty acid composition of cocoa butter is typically 26 percent palmitate, 34 percent stearate, 35 percent oleate and 3 percent linoleate. The unique fatty acid composition of cocoa butter confers properties that make this edible fat eminently suitable for confectionery end-uses. Cocoa butter is hard and non-greasy at ordinary temperatures, and melts very sharply in the mouth. It is extremely resistant to oxidative reactions. For these reasons, producing sunflower oil with increased levels of stearate and palmitate, and reduced levels of unsaturated fatty acids could expand the use of sunflower as a cocoa butter substitute. Such a replacement of cocoa butter with sunflower oil would provide value to oil and food processors as well as reduce the foreign import of tropical oils.
Other traditional uses of saturated fat, such as raw material for the manufacture of soap and the coating of foods, could be filled by a sunflower oil having increased levels of stearate and palmitate. Animal fat is employed for these purposes because it contains a high level of saturated fatty acids. This gives animal fat a greater resistance to oxidation. Sunflower oil having increased levels of stearate and palmitate would similarly be less resistant to oxidation.
Coupled with traditional breeding, mutagenesis has been used to create sunflower varieties that have altered fatty acid compositions. One example of a sunflower variety made this way is Pioneer.RTM. hybrid 6661, which produces a seed storage oil having a fatty acid composition of about 85 percent oleate. Another example is a sunflower variety that bears seeds with a high stearate content as described by Osorio et al., Crop Sci. 35: 739-42 (1995).
The mutagenesis approach is severely limited in this context, however. It is unlikely to create a mutated variety in which a dominant or "gain-of-function" phenotype is created by a gene that is essential for plant growth or by a gene that exists in more than one copy. Also, slow and expensive traditional breeding techniques are required to introgress a mutation into an elite line. This problem stems from polygenic inheritance of genes that cause the desired oil composition.
The polygenic inheritance problem is evident in the high stearate lines created by Osorio et al. (1995), supra, in which seed stearate compositions changed through several generations. After three generations of inbreeding, sunflower seeds from two lines reported by Osorio et al. contained more than 10 percent stearate, less than 6 percent palmitate, and more than 13 percent oleate.
Recent molecular and cellular biology techniques offer the prospect of overcoming some of the limitations of the mutagenesis approach, including the need for extensive breeding. Particularly useful technologies are (a) seed-specific expression of foreign genes in transgenic plants, (b) use of antisense RNA to inhibit plant target genes in a dominant and tissue-specific manner, (c) transfer of foreign genes into elite commercial varieties of commercial oil crops such as sunflower, as described by Everett et al., Bio/Technology 5: 1201-04 (1987), and (d) use of genes as restriction fragment length polymorphism markers in a breeding program, which makes introgression of recessive traits into elite lines rapid and less expensive, as described by Tanksley et al., Bio/Technology 7: 257-64 (1989). But each of these technologies requires the identification and isolation of commercially important genes.
Some commercially important genes involved in fatty acid synthesis within the plant have been identified. For example, the biosyntheses of palmitate, stearate and oleate occur in the plastids by the interplay of three key "ACP track" enzymes: palmitoyl-ACP elongase, stearoyl-ACP desaturase and acyl-ACP thioesterase. Stearoyl-ACP desaturase introduces the first double bond on stearoyl-ACP to form oleoyl-ACP. This enzyme is pivotal and determines the degree of eighteen carbon length fatty acid unsaturation in vegetable oils.
Fatty acids synthesized in the plastid are exported as acyl-CoA to the cytoplasm. At least three different glycerol acylating enzymes: glycerol-3-P acyltransferase; 1-acyl-glycerol-3-P acyltransferase; and diacylglycerol acyltransferase incorporate acyl moieties from the cytoplasm into triglycerides during oil biosynthesis. These acyltransferases show a strong, but not absolute, preference for incorporating saturated fatty acids at positions 1 and 3 and monounsaturated fatty acid at position 2 of the triglyceride. Thus, altering the fatty acid composition of the acyl pool can drive by mass action, a corresponding change in the fatty acid composition of the oil. Furthermore, there is experimental evidence that, because of this specificity, given the correct composition of fatty acids, plants can produce cocoa butter substitutes. Bafor et al., JAOCS 67: 217-25 (1990).
Thus, one approach to alter the levels of stearate and oleate in sunflower oil entails modifying the levels of cytoplasmic acyl-CoA. This can be done genetically in two ways. First, altering the biosynthesis of stearate and oleate in the plastid may be effected by modulating the levels of stearoyl-ACP desaturase in seeds, either through overexpression or antisense inhibition of the stearoyl-ACP desaturase gene. A second approach involves converting stearoyl-CoA to oleoyl-CoA in the cytoplasm through the expression of stearoyl-ACP desaturase in the cytoplasm.
Antisense inhibition of a stearoyl-ACP gene can be achieved by placing a DNA segment in a cell such that it produces an RNA that is complementary to the stearoyl-ACP desaturase mRNA. For this strategy, a stearoyl-ACP desaturase gene is first isolated from which to make the DNA segment. It is preferred to isolate the actual gene(s) or cDNA(s) encoding stearoyl-ACP desaturase from sunflower, and not from another species. This is because antisense inhibition works best when there is a high-degree of complementarity between the antisense RNA and the targeted gene.
Antisense inhibition of plant stearoyl-ACP desaturase in canola has been reported by Knutzon et al., Proc. Nat'l Acad. Sci. USA 89: 2624-28 (1992); see also U.S. Pat. No. 5,443,974. These publications describe an increased level of stearate produced by antisense inhibition in seeds of canola and soybean, respectively. In the case of canola, transgenic seeds having the highest levels of stearate contained about 15 percent oleate, a four-fold reduction from the control value of about 60 percent oleate. In the case of soybean, the increased level of stearate was not accompanied by a decreased level of oleate. In both instances a cloned stearoyl-ACP desaturase from the respective plant species was used to construct transgenic DNA segments.
The expectation that antisense expression of the stearoyl-ACP desaturase gene only would affect the concentration of 18-carbon fatty acids was borne out by the data of Knutzon et al. (1992), supra, who found that an increase in stearate was not accompanied by an increase in palmitate. The experimental data set out in U.S. Pat. No. 5,443,974 also reveal an increase in stearate unaccompanied by an increase in palmitate.
Thus, the genetic alteration of plant stearoyl-ACP desaturase can affect the levels of 18-carbon fatty acids, but is not known to influence 16-carbon fatty acid metabolism. Yet an increase in both stearate and palmitate is desired for certain commercial uses, as summarized above. Accordingly, an approach to achieve a vegetable oil with a high stearate content and a high palmitate content would be a valuable addition to the art.